Biodegradability of antineoplastic compounds in screening tests: influence of glucosidation and of stereochemistry
Introduction
Only a few data on the presence of pharmaceuticals in surface water and in the effluent of sewage treatment plants were reported in the eighties (Richardson and Bowron, 1985). More recently, pharmaceuticals have been detected in ground water and drinking water (Stan et al., 1994). However, only little is known about fate and effect of pharmaceuticals and their metabolites in the environment (Halling-Sørensen et al., 1998). After administration, pharmaceuticals are often metabolised. The degree of metabolism depends on several parameters, such as the constitution of the patient or the time of administration Touitu and Haus, 1992, Lemmer, 1996 and some part of the drug is excreted unchanged.
Cyclophosphamide (CP) and ifosfamide (IF), both isomeric alkylating N-lost derivatives (Fig. 1), are among the most frequently used antineoplastics in the clinic. They are supposed to be mutagenic, carcinogenic, teratogenic, and embryotoxic IARC, 1975, IARC, 1981, Allwood and Wright, 1993, ASTA Medica, 1995. Data for acute toxicity are available (e.g. ASTA Medica, 1995): rat, oral (OECD 401) LD50=379 mg/kg (female) and 568 mg/kg (male).
The active metabolite of IF and CP is isophosphoramidmustard (IPM) which is highly reactive and is therefore not used therapeutically. β-d-Glc-IPM and β-l-Glc-IPM (Fig. 2) were synthesised only recently. The conjugation of the IPM as an aglycon to the reducing end of glucose has resulted in new compounds. The new drug β-d-Glc-IPM as well as β-l-Glc-IPM is in contrast to the agylcon isophosphoramidmustard chemically stable. Only β-d-Glc-IPM is active against tumours Pohl et al., 1995, Stüben et al., 1996 and has major advantages. An improvement of the pharmacological properties compared with IF is expected. At present, β-d-Glc-IPM is being investigated by ASTA Medica as antineoplastic substance in clinical trials (INN=glufosfamide). Both compounds β-d- and β-l-Glc-IPM belong to the group of nitrogen mustard-derived alkylating agents. CP and IF are the best known representatives of this class and play an important role in chemotherapy.
For IF high chemical stability up to 40°C and pH between 4.5 and 9 was found; only 10% of IF was hydrolysed within 94±1.4 days at 25°C and pH 7 (Muñoz et al., 1996). IF and CP like several other investigated antineoplastics were not biodegradable in different tests Kümmerer et al., 1997, Steger-Hartmann et al., 1997. Both CP and IF were detected in the influent of a municipal sewage treatment plant in nearly the same concentration as in the effluent, indicating that no elimination took place. CP and IF are emitted unchanged into the surface water. Effluent concentrations of a communal STP ranged from <10 to 3 μg/l (peak concentration during the day time) as reported by Steger-Hartmann et al., 1996, Steger-Hartmann et al., 1997, Kümmerer et al., 1997 and Kümmerer (1998). Therefore IF and CP have to be assumed to be persistent in the aquatic environment.
The environmental fate of chemicals partly depends on their stereochemistry (Kohler et al., 1997). IF, like other oxazaphosphorine drugs, is chiral. The pharmacokinetics of racemic ifosfamide, R-ifosfamide and S-ifosfamide, in humans was studied only recently. There is some evidence from animal studies and in humans of stereo-selective differences in metabolism, excretion and cytotoxic activity between the two enantiomers (Boos et al., 1991). The clearance of S-IF in children was higher than that of R-IF (Prasad and Lewi, 1994).
Biodegradation of organic compounds in the environment e.g. by bacteria occurs catalysed by more or less stereo-specific enzymes. In laboratory testing only little biodegradation (Kümmerer et al., 1996a) and elimination in laboratory scale sewage treatment plants of racemic CP and IF has been found Kümmerer et al., 1997, Steger-Hartmann et al., 1997 indicating that there was no stereo-specific elimination. Under anaerobic conditions, 50% elimination was found for racemic ifosfamide (Schecker et al., 1998), but stereo-chemical analysis was not performed. β-d-Glc-IPM can be split by intracellular glycolytic enzymes. β-l-Glc-IPM and β-d-Glc-IPM are enantiomers.
Therefore, we investigated the biodegradability of β-d-Glc-IPM and compared it with the biodegradability of β-l-Glc-IPM to compare the biodegradability of two enantiomers. Standard test systems were used as recommended by the OECD for testing of chemicals (OECD, 1992). The closed bottle test (CBT) (OECD 301 D) was used as a test with low bacterial density for ready biodegradability (level 0 screening test) and the Zahn–Wellens test (ZWT) (OECD 302B) was used for assessing the biodegradability at high bacterial density in a batch test (level 1 screening test). Before biodegradability testing growth inhibition tests (GITs) with Pseudomonas putida were performed to exclude toxicity of the test compounds against test organisms. An influence of the test compounds on the bacteria in the test vessels was also assessed using toxicity controls and by counting the colony forming units.
Section snippets
Growth inhibition test
GITs were conducted in duplicates, according to an international method (ISO, 1995). Eight fold doubling concentrations were used, starting at an initial concentration of 2 mg/l for both test compounds. Several modifications were made, as described elsewhere in detail (Kümmerer et al., 1996b). The disinfectant benzalkonium chloride was used at a concentration of 32 mg/l as control for 100% inhibition. The incubation temperature was 30°C. The nutrient solution was adapted to waste water
Toxicity against waste water bacteria
For both β-d- and β-l-Glc-IPM the inhibition concentration against Ps. putida was above 256 mg/l. In the toxicity controls of the CBT and the ZWT no inhibition was observed according to test guidelines. The monitoring of the CFUs showed that the cultivable bacteria were not affected by the test compounds.
Biodegradability
β-d-Glc-IPM (Fig. 3) was more biodegradable in the CBT than its enantiomer isomer β-l-Glc-IPM (Fig. 4). None of the test compounds reached the level necessary for classification as “readily
Discussion
The fate and effects of pharmaceuticals in the aquatic environment have gained increasing scientific interest in the last years. Although a lot of compounds have been detected in surface waters, only little information on the environmental fate of pharmaceuticals in the aquatic environment is available (Halling-Sørensen et al., 1998). Persistence (Klöpffer, 1989, Kümmerer and Held, 1997) of a compound is an important criterion for risk assessment. Only one antineoplastic compound out of 12
Conclusions
Quite a lot of pharmaceuticals have been detected in the aquatic environment. Therefore, in the development of new active substances better biodegradability in the environment should be considered more than in the past. Glucosidation of IPM resulted not only in less side effects for patients but also better environmental biodegradability. Biodegradabily in sewage treatment plants or surface water may differ from the obtained results due to other possible processes of elimination of the
Acknowledgements
The authors appreciate the excellent technical assistance of I. Bulowski (Zahn–Wellens tests) and R. Welte (growth inhibition tests).
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